Capillary flow device

ABSTRACT

Novel methods and devices are provided involving at least one chamber, at least one capillary, and at least one reagent involved in a system providing for a detectable signal. As appropriate, the devices provide for measuring a sample, mixing the sample with reagents, defining a flow path, and reading the result. Of particular interest is the use of combinations of specific binding pair members which result in agglutination information, where the resulting agglutination particles may provide for changes in flow rate, light patterns of a flowing medium, or light absorption or scattering. A fabrication technique particularly suited for forming internal chambers in plastic devices is also described along with various control devices for use with the basic device.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 177,625, filed Apr. 5, 1988;which is a division of application Ser. No. 880,793, filed July 1, 1986;which is a continuation-in-part of application Ser. No. 762,748, filedAug. 5, 1985.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is related to testing devices having internal chambersinto which fluids are drawn by capillary action, to methods of usingsuch devices, and to methods of manufacturing such devices.

2. Background of the Invention

In the development of the diagnostics field, there has been explosivegrowth in the number of substances to be determined. For the most part,the medical field has looked to clinical laboratories for thesedeterminations. The clinical laboratories have been dependent uponexpensive sophisticated equipment and highly trained technical help tofulfill the manifold needs of the medical community. However, in ahighly automated clinical laboratory, there is substantial need toperform one or a few assays on a stat basis with minimum equipment.

There is also an expanding need for having analytical capabilities indoctors' offices and in the home. There is a continuing need to monitorthe level of drug administered to people with chronic illnesses, such asdiabetics, asthmatics, epileptics, and cardiac patients, as it appearsin a physiological fluid, such as blood. Tests of interest includeprothrombin time, potassium ion, and cholesterol. Determiningred-blood-cell count is also a common test. In the case of diabeticpatients, it is necessary to determine sugar level in urine or blood.

Numerous approaches have been developed toward this end, depending tovarying degrees on instrumental or visual observation of the result.Typical of these are the so called "dip-stick" methods. These methodsgenerally employ a plastic strip with a reagent-containing matrixlayered thereon. Sample is applied to the strip and the presence orabsence of a analyte is indicated by a color-forming reaction. Whilesuch devices have proven useful for the qualitative determination of thepresence of analytes in urine and can even be used for roughquantitative analysis, they are not particularly useful with whole bloodbecause of the interferring effects of red blood cells, nor are theyuseful for making fine quantitative distinctions. Accordingly, thereremains a need for the development of methods and devices capable ofanalyzing whole blood and other complex samples rapidly with a minimumof user manipulations.

Many small devices in the analytical area depend on the use of plasticshaving specified characteristics, such as optical transparency andmachinability. Machinability refers here to the ability to producechambers, channels, and openings of prescribed dimensions within theplastic device. Although numerous plastic devices have been devised, thefabrication techniques are not interchangeable because of differences inthe devices or the desired measuring result. This is particularly truefor devices containing channels or other chambers of small dimensionsinternally in the plastic material. The fine channels are difficult toproduce entirely within a plastic matrix and, if prepared in the surfaceof two matrices to be sealed to each other, are readily deformed duringmany sealing processes.

Accordingly, there remains a need for new devices for use in methods ofrapid analytical testing and for new methods of producing these devices.

BRIEF DESCRIPTION OF THE RELEVANT LITERATURE

Powers et al., IEEE Trans. on Biomedical Engr. (1983) BME-30-228,describes detecting a speckle pattern for determining plateletaggregation, as does Reynolds, Light Scattering Detection ofThromboemboli, Trans. 11th Annual Mtg. of the Soc. for Biomaterials, SanDiego, Calif., Apr. 25-28, 1985. Reynolds and Simon, Transfusion (1980)20:669-677, describes size distribution measurements of microaggregatesin stored blood. Of interest in the same area are U.S. Pat. Nos.2,616,796; 3,810,010; 3,915,652; 4,040,742; 4,091,802; and 4,142,796.U.S. Pat. No. 4,519,239 describes an apparatus for determining flowshear stress of suspensions in blood. Ab Leo sells the HemoCue™ devicefor measuring hemoglobin. Also, see U.S. Pat. No. 4,088,448, whichdescribes a cuvette for sampling with a cavity which is defined in sucha manner as to draw into the cavity a sample in an amount which isexactly determined in relation to the volume of the cavity by capillaryforce. Numerous plastic assembly techniques, particularly ultrasonicsplastic assembly, is described in a book of the same name published byBranson Sonic Power Company, Danbury, Conn., 1979. Gallagan, PlasticsEngineering, Aug. 1985, 35-37 also describes ultrasonic welding ofplastics. U.S. Pat. No. 3,376,208 describes corona discharge, althoughfor a different purpose. U.S. Pat. No. 3,376,208 describes the use of anelectric discharge to modify a film surface. A device used to transportliquids by capillary flow is described in U.S. Pat. No. 4,233,029.

SUMMARY OF THE INVENTION

The present invention provides fabrication techniques, the resultingdevices, and techniques related to the use of such devices in which adefined chamber or channel is prepared within the internal space of asolid device. The devices typically call for the use of capillary forceto draw a sample into the internal chambers of a plastic device. Suchcapillary flow devices, particularly capillary flow devices designed fora constant flow rate, typically include at least one capillary acting asa pump, usually for controlling the volume of the sample and the timeperiod for reaction, a chamber, an inlet port, a vent, and a reagent inproximity to at least one surface of the device. The capillary andchamber provide for capillary flow due to surface action and for mixingof the assay medium with the reagent. The reagent is part of a detectionsystem, whereby a detectable result occurs in relation to the presenceof an analyte. The device and the corresponding method can be used witha wide variety of fluids, particularly physiological fluids, fordetection of drugs, pathogens, materials endogenous to a host, or thelike. In most cases an optical measurement is being made, which requiresthe selection of a transparent material. Devices of unusuallyadvantageous properties can be prepared by injection moldingacrylonitrile-butadiene-styrene copolymer (ABS) so as to form adepression of defined dimensions in the surface of at least one face ofthe polymer, increasing wettability of the surface in at least thoseportions defined by the depression using either plasma etching or coronadischarge, providing energy directing ridges projecting from the surfaceof the plastic adjacent to the depression or in a second piece ofplastic so shaped as to contact the area adjacent to the depressions inthe plastic surface, and ultrasonically welding the two plastic surfacesso as to produce an internal chamber or channel of defined dimensionshaving an air-tight seal around the perimeter of the resulting chamber.Although the fabrication method can be used to produce internal chambersof any dimension, the method is particularly suitable for the productionof chambers and channels of small dimensions that are suitable forinducing capillary flow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are plan views of two embodiments of the subjectinvention, FIG. 1A employing a single capillary and chamber and FIG. 1Bemploying two capillaries separated by a chamber.

FIGS. 2A and 2B are a plan view and a side elevational view of a deviceemploying three chambers.

FIG. 3 is a plan view of a device for a plurality of simultaneousdeterminations.

FIG. 4 is a plan view of an alternate embodiment, where the sample isdivided into two separate channels.

FIG. 5 is a plan view of an alternate embodiment employing an extendedcapillary path.

FIG. 6 is a cross-sectional view of an embodiment showing the locationof channels and energy directing ridges during the fabrication process.

FIG. 7 is a block diagram of an electronic circuit suitable for use inan electronic capillary cartidge device to simulate the passage of bloodthrough a capillary in a control cycle.

FIG. 8 is a diagram of the physical location and electronic circuitry ofa detector capable of determining depletion of sample in a reservoir ofa capillary device.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

This invention provides devices and methods, where the devices rely uponcapillaries, chambers, and orifices to pump fluids; to controlmeasurement of fluids, reaction times, and mixing of reagents; and todetermine a detectable signal. By varying the path through which thefluid flows, one can provide for a variety of activities, such asmixing, incubating and detecting. The methods may involve the binding ofhomologous members of a specific binding pair, resulting in complexformation. The complex formation can provide for a variety of eventswhich can be detected by instrumentation or visual means. Alternatively,the methods may involve chemical reactions, for example, the detectionof glucose or serum enzymes, which result in a detectable change in thesample medium. Since the devices rely upon capillaries and otherchambers to control movement of fluids, accurate control of thedimensions of the internal chambers is essential. The fabricationtechniques described later provide this accurate control.

The sample may be a fluid which is used directly as obtained from thesource or may be pretreated in a variety of ways so as to modify itscharacter. The sample will then be introduced into the device through aninlet port, which may introduce the sample into a chamber or acapillary. The sample will then transit the device passing through thecapillary(ies) or chamber(s), where the sample will encounter one ormore reagents, which reagents are involved in a system which produces adetectable signal. By having orifices which connect the pathway to theatmosphere at one or more sites, one can terminate the flow up to thatsite, so that the medium may be incubated for various times or movementstopped subject to the initiating movement, for example, immediatelyprior to measurement.

Any liquid sample may be employed, where the sample will have areasonable rate of flow due to the pumping of the capillary action. Itis to be understood the capillary action is the sole driving force,relying on the surface action between the surface and the fluid. Wherethe sample is too viscous, it should be diluted to provide for acapillary pumping rate which allows for the desired manipulations, suchas mixing, and for a reasonable flow time which will control the timeperiod for the assay.

The sample may be derived from any source, such as a physiologicalfluid, e.g., blood, saliva, ocular lens fluid, cerebral spinal fluid,pus, sweat, exudate, urine, milk, or the like. The fluid may besubjected to prior treatment, such as preparing serum from blood,diluting pus, saliva, or the like; the methods of treatment may involveconcentration, by filtration, distillation, dialysis, or the like;dilution, filtration, inactivation of natural components, concentration,chromatography, addition of reagents, chemical treatment, etc.

Besides physiological fluids, other liquid samples may be employed wherethe component(s) of interest may be either liquids or solids and thesolid(s) dissolved in a liquid medium. Samples of interest includeprocess streams, water, soil, plants or other vegetation, air, and thelike.

The analytes of interest are widely varied depending upon the purpose ofthe assay and the source. Analytes may include small organic molecules,such as drugs, hormones, steroids, neurotransmitters, growth factors,commercial chemicals, degradation products, drugs of abuse, metabolites,catabolites, etc. Large organic molecules may be determined, such asnucleic acids, proteins, polysaccharides, or the like. Aggregations ofmolecules may also be of interest, particularly naturally-occurringaggregations such as viroids, viruses, cells, both prokaryotic andeukaryotic, including unicellular microorganisms, mammalian cells suchas lymphocytes, epithelial cells, neoplastic cells, and the like.

Phenomena of interest which may be measured may be indicative ofphysiological or non-physiological processes, such as blood clotting,platelet aggregation, complement-mediated lysis, polymerization,agglutination, etc.

The sample medium may be the naturally-occurring medium or the sampleintroduced into a liquid meidum which provides for the desiredcharacteristics necessary for the capillary pumping action and thedetectable signal. For the most part, aqueous media will be employed andto that extent aqueous media will be exemplary of the media employed inthe subject invention. The aqueous media may be modified by the additionof a variety of miscible liquids, particularly oxygenated organicsolvents, such as lower alkanols, dimethyl formamide, dimethylsulfoxide, acetone, or the like. Usually, the solvents will be presentin less than about 40 volume percent, more usually in less than about 20volume percent. Besides other solvents, other liquid or solid additivesmay be included in the medium to modify the flow or other properties ofthe medium, such as sugars, polyols, polymers, detergents, surfactantsand the like, involved with changes in wetting, adherence, laminar flow,viscosity, and the like.

In addition to the components mentioned above, other additives may beincluded for specific purposes. Buffers may be desirable to maintain aparticular pH. Enzyme inhibitors may be included. Other reagents ofinterest are antibodies, preservatives, stabilizers, activators, enzymesubstrates and cofactors, oxidants, reductants, etc. In addition,filtration or trapping devices may be included in the device pathway, soas to remove particles above a certain size. The particles may includecells, viruses, latex particles, high molecular weight polymers, nucleicacids, by themselves or in combination with proteins, e.g., nucleosomes,magnetic particles, ligand or receptor containing particles, or thelike.

The sample may provide the detectable component of the detection systemor such component may be added. The component(s) will vary widelydepending upon the nature of the detection system. One detection methodwill involve the use of particles, where particles provide for lightscatter or a change in the rate of flow. The particles may be cells,polymeric particles which are immiscible with the liquid system, latexparticles, charcoal particles, metal particles, polysaccharides orprotein particles, ceramic particles, nucleic acid particles,agglutinated particles, or the like. The choice of particle will dependupon the ease of detection, the dispersability or stability of thedispersion, inertness, participation in the change in flow, and thelike. Particle sizes will generally be from about 0.1-100μ, more usuallyfrom about 5-15μ. Other phenomena which may be detected include changesin color, light absorption or transmission, fluorescence, change inphysical phase, or the like.

The neat sample or formulated sample will be introduced into the entryport into the receiving unit of the device. The receiving unit may be acapillary or a chamber. The receiving unit may be used to measure theparticular sample volume or may simply serve to receive the sample anddirect the sample to the next unit of the device. The capillary unitsserve a variety of functions, including a measuring device for volumemeasurement, a metering pump for transferring liquid from one chamber toanother, flow controller for controlling the rate of flow betweenchambers, mixer for mixing reagents, and detecting unit for detection.For the most part, the capillaries will serve as transfer units, flowcontrol units and detection units. The chambers and capillaries may beused to define different events, e.g., areas of reaction, or differentstructural entities in certain embodiments of the method.

The capillaries will usually be of a substantially smaller cross-sectionor diameter in the direction transverse to the direction of flow thanthe chambers. The cross-section or length in the direction of flow maybe similar or may differ by a factor of ten or more depending on thefunction of the capillary and the chamber. Capillaries will usually havediameters in the range of about 0.01 mm to 2 mm, usually about 0.1 mm to1 mm. The length of the capillary, particularly the first capillary inthe pathway, more particularly the first capillary when it is joined tothe entry port, will be at least about 3 mm, more usually at least about5 mm, and may be 1 cm or more, usually not more than about 2 cm, whilesubsequent capillaries may be shorter or longer, frequently at least onebeing longer, being as long as 10 cm, usually not exceeding about 5 cm.

The first capillary will initially control the rate of flow into thechamber which will usually serve as the reaction chamber. Thus, thecapillary may aid in the control of the time with which the assay mediumis in contact with reagent contained within or bound to the walls of thecapillary and/or reaction chamber and the progress of the assay mediumthrough the chamber. Other components which may affect the rate of flowin the chamber include baffles, walls or other impedimenta in thechamber, the geometry of the chamber, the reagent in the chamber and thenature of the surfaces in the capillary and chamber. Since in manyinstances the initial contacting of the assay medium and the reagentcould affect the results, it is desirable that the contact besufficiently slow that equilibrium can occur, as to dissolution,reaction, etc.

The capillary control and use of relatively thin heat conductive wallsallows for rapid heat transfer and isothermal conditions, oralternatively, thick walls can provide for adiabatic conditions. Thus,the small volume of fluid in the chambers and capillaries permits forrapid heat exchange or efficient thermal insulation. In addition, thethin capillaries permit optical measurements, particularly based ontransmission of light, with optically dense samples, e.g., whole blood.There is the further opportunity for rapid efficient mixing, where bysonication the whole sample can be uniformly mixed.

The capillary provides the sole driving source for the movement ofliquid through the device. Accordingly, careful fabrication of thecapillary to exact dimensions is required. The device is normallyemployed in the horizontal position, so that gravity does not affect theflow rate. The composition of the walls of the capillary are selected soas to provide the desired degree of wetting and surface tension or thewalls are modified to provide the desired physical properties. Thedevice is employed without ancillary motive force, such as pumps,gravity or the like.

The chambers also have a variety of functions, serving as protection forthe reagent(s) mixing chambers for dissolution of reagent and/orreaction with reagent, volume measurement, incubation and detection,where the detectable signal is other than a signal associated with flow,and the like. The chambers will be primarily employed for mixing,incubating and for holding of liquids.

Depending upon the particular system, the length of the capillaries,their cross-sectional area, the volumes of the various chambers, andtheir length and shape, may be varied widely. One constraint on each ofthe capillaries is the necessity for their function providing capillarypumping action for flow. Therefore air leaks in the space surroundingthe capillary (except for designed access ports) cannot be tolerated. Inmany instances, the chambers will also provide for capillary actionwhile the flow rate which will be affected by the nature of thecapillary surface will be primarily determined by the capillary actionof the capillaries.

In order to minimize the handling of reagents by the user of the device,the reagents may be supplied within the device, where by mixing with thereagents occurs in the device. The reagents may be present eitherdiffusively or non-diffusively bound to the surface of the device, thatis, adhered, absorbed, absorbed or covalently-linked, so that thereagent may become dissolved in the fluid or may remain fixed to thesurface. Where the reagents are diffusively bound (non-covalently andweakly bound), a variety of situations can be accommodated. Onesituation is where the liquid front dissolves all of the reagent, sothat the liquid front receives a high concentration of the reagent andmost of the reaction occurs at the liquid front. A second situationwould be with an excess of a reagent of limited solubility. In thissituation, the reagent may be present in the liquid medium at asubstantially uniform concentration. A third situation is to have adeficiency of a reagent of limited solubility, so that only the earlyportion of the fluid will have a relatively constant reagentconcentration. In many instances it is essential that the reagent bepresent in a defined area or reaction chamber, which makes fabricationof an internal chamber followed by later addition of reagent virtuallyimpossible.

While for the most part, the reagent will be present in one or moreunits of the device, reagents also can be mechanically introduced byvarious techniques. For example, by employing a septum, a syringe may beemployed for introducing a reagent. Alternatively, one could have anorifice and use an eyedropper or other means for introducing liquidreagent into the unit. Usually, unless essential, these alternativetechniques will be avoided.

The reagent will vary depending upon the nature of the sample, theanalyte, and the manner in which the detectable signal is generated. Achemical reaction will occur due either to the formation of covalentbonds, e.g., oxidation or reduction, hydrolysis, etc., or non-covalentbonds, e.g., complex formation between ligand and receptor, includingcomplex formation between nucleic acids.

The same or different reagent may be present in the various units, sothat successive reactions can occur or a reagent continually suppliedinto the moving medium. Also, one could have a plurality of chambers andcapillary channels. Frequently, the first unit will have a reactant. Thechambers can be varied in size and purpose, providing the varyingincubation times, varying reaction times, mixing of media from differentcapillaries, or the like. Any number of chambers may be employed,usually not exceeding six, more usually not exceeding about four, wherethe chambers may be in series or parallel. The size of the unit, eithercapillary or chamber, can be particularly important, where the reagentis fixed, so that the residence time in contact with the reagent will beaffected by the area of the reagent contacted by the assay medium.

By employing various filtration or trapping devices (e.g., mechanical ormagnetic), one can inhibit the transfer of particles from a capillarychannel to a chamber or vice versa. In this way, red cells can beremoved from blood, various components of the sample may be removed, orby employing divergent channels, one channel can have particles removedand the particles retained in the other channel where the two resultsmay be of interest.

Arbitrarily, the use of the device will be divided into two differentconcepts. The first concept will involve a characteristic other than achange in flow rate. For the most part, this will involve theabsorption, scatter or emission of light. A wide variety of protocolsand reagents are available which provide for a change in the measuredlight, as a result of absorption, scatter or emission, in relation tothe amount of analyte in the sample.

Labels which may be employed include enzymes, in combination withsubstrates, cofactors or inhibitors, fluorescers, combinations offluorescers and quenchers, dyes, or the like. In some instances achemical reaction occurs as a result of the presence of the analyte orwith the analyte, which provides a detectable signal. By employingappropriate protocols, the amount of absorption or emission of light inthe detection unit can be directly related to the amount of analyte inthe sample.

Detection of a change in the rate of flow may be the signal whichresults from the reaction of the label or may be the result of acombination of a plurality of entities, which affect the rate of flow.The change in flow rate may be as a result of agglutination,polymerization, complex formation between high molecular weightcompounds or aggregations, or the like.

The measurement of light, e.g., scatter, can be used to measure a changein the size population. This can be particularly useful for measurementof agglutination, clumping, clot formation or dissolution, and the like.A laser is able to distinguish particle size without a change in theflow rate. Small particles have a low frequency and a high amplitude;large particles (agglutinated particles) have a lower frequency (fewertotal particles) and a higher amplitude (each particle is larger). Thusthe change in particle size distribution may be detected by integratednoise employing known circuitry.

Various situations can be involved where the assay medium may have noparticles (entities capable of scattering) or have particles, such ascells, latex beads and the like, where the result of the reaction is tochange the particle size distribution, including going from no particlesto the formation of particles. There is also the opportunity to beginwith particles, e.g., blood clots, and as a result of the reactionreduce the size and number of particles, e.g., dissolve the blood clots.

Protocols which may find use include those found in U.S. Pat. Nos.3,817,837; 3,839,153; 3,998,943; 3,935,074; 4,174,834; 4,233,402;4,208,479; 4,235,869; 4,275,149; 4,287,300, etc., whose relevantdisclosure is incorporated herein by reference.

Because of the enormous diversity of protocols which are presentlyavailable which can be employed in the subject methods and devices, onlya few will be illustrative and reference will be made to numerouspatents which describe different protocols.

In a first exemplary protocol, a fluorescence measurement may be made,where one has a single capillary as the inlet, with the capillary coatedwith antibody to analyte. The sample is diluted with a buffered reagentcontaining a conjugate of analyte with fluorescer, whereby all of thefluorescent reagent will become bound in the capillary in the absence ofany analyte in the sample. The capillary is then introduced into thesample and an amount of liquid withdrawn up to an indexed point on thecapillary, whereby the capillary is then withdrawn from the sample andthe liquid allowed to progress into the chamber. When the chamber ispartially or completely full, the fluorescence of the chamber may thenbe read as indicative of the amount of analyte in the sample.

With enzymes, one could either vary the protocol or the device toprevent premature interaction between the enzyme and its substrate orinhibitor. Where a simple two-unit device is employed, employing acapillary and chamber, one could provide for using a combination ofenzymes, referred to as channeling, where the product of one enzyme isthe substrate of the other enzyme. In this manner, one could have in oneunit a second enzyme combined with the substrate of the first enzyme,while the second unit having the first enzyme combined with thesubstrate of the second enzyme.

One could modulate the reaction by various means. For example, one couldhave antibody to analyte in the first unit and combine the sample with abuffered solution of antibody-first enzyme inhibitor conjugate. Thus,the amount of enzyme inhibitor which would enter the second unit wouldbe related to the amount of analyte in the sample. Instead of having atwo-unit system, one could have a three-unit system, where the firstunit mixes the sample with the enzyme inhibitor conjugate avoiding thenecessity to combine the sample with a liquid medium. Where the sampleis colored, such as blood, it may be necessary to filter out or trap redblood cells, to allow for development of color or fluorescence or tofind a wavelength range, where one can read the development of aparticular light-absorbing material.

By employing a plurality of units, one can use a single enzyme, wherethe enzyme is conjugated to the analyte. By having enzyme-analyteconjugate in a first unit, followed by antibody to analyte in a secondunit and employing a third unit containing enzyme substrate as thereaction chamber, the measurement can be made in the third unit.

By employing combinations of filters and particles one could alsoachieve similar effects. For example, one can employ enzyme analyteconjugates in the first unit which completely dissolve in the assaymedium. A second unit may then contain the particles containing antibodyto the analyte. The amount of enzyme-analyte conjugate which binds tothe antibodies will be dependent upon the amount of analyte in thesample. By having a filter at the exit of the second unit, all of theparticles will be trapped at the filter, and only enzyme conjugate whichis unbound will pass through the filter to the third unit. The thirdunit may then contain the enzyme substrate, so that reaction of theenzyme with the substrate can be monitored.

For the detection of change in flow rate, a wide variety of systems maybe employed. Of particular interest is the natural system involvingclotting, which is convenient when the sample is a blood sample. Thus,by adding one or more components of the clotting cascade or a componentwhich activates the naturally-occuring components present, the clottingcan provide for a change in the flow rate. Particularly, these reagentsmay include thromboplastin, factors I, II, IV, V, VII, VIII, IX, X, XIand XII. These components can be added individually or in combination.Particular combinations include factors of the intrinsic pathway (VIII,IX, XI and XII) or extrinsic pathway (III, VII) with the common pathwayfactors I, II, V, X, XIII. The clotting assay can be used to determine awide variety of analytes of interest. The clotting assay may be used forthe detection of the presence of anti-clotting agents or clottingagents. In addition, the clotting assay can be used for the detection ofthe level of a particular component required for clotting or a componentinvolved in the dissolving of clots. Illustrative analytes include thevarious factors indicated above, warfarin, tissue plasminogen activator,heparin, streptokinase, Vitamin K, anti-platelet drugs, protaminesulfate, etc.

The clotting assay can also be used to measure any analyte of interestby having the analyte associated with a factor necessary for clotting.For example, by conjugating thromboplastin to a compound capable ofcompeting with the analyte for a receptor, where thethromboplastin-receptor complex is inactive, one could determine thepresence of the analyte. The sample would be mixed with the receptor forthe analyte so that the binding sites of the receptor would be occupiedby any analyte present. The thromboplastin conjugated to the analyte ormimetic analog would be the reagent in the reaction chamber. All theother components necessary for clotting would be included in the samplealong with the antibodies for the analyte, where such other componentswere not naturally present in sufficient amount in the sample. Thereceptor sites which were not occupied by analyte would bind to theanalyte conjugated to the thromboplastin where the resulting specificbinding complex would lack thromboplastin activity. The remaininguncomplexed thromboplastin conjugate would be active and initiateclotting. By employing appropriate standards, one could relate the timeto flow stoppage to the amount of analyte in the sample. Or, one couldprovide that flow stoppage did or did not occur with an amount ofanalyte above a threshold level.

Besides coagulation, agglutination, precipitation, or plug formation byother means, or, as appropriate, increasing viscosity can be used aspart of the detection scheme.

For plug formation or slowing of flow by other than clotting, particleswill usually be included in the medium which become cross-linked inrelation to the amount of analyte present. This can be achived byemploying receptors and ligands specific for the receptor so thatnon-covalent binding provides for multiple linkages between particleswith a resulting cross-link structure which can serve as a plug. Byusing such particles as S. aureus, which specifically binds to immunecomplexes, red blood cells, synthetic or naturally-occurring particlesto which ligands, rheumatoid factor, antibodies, naturally-occurringreceptors, or the like, are conjugated, the cross-linking of theparticles can be retarded or enhanced by the presence or absence of theanalyte. Thus, in the case of antigens, the antigen may serve as abridge between different antibodies, while in the case of haptens,individual haptens may inhibit the cross-linking resulting from apolyhaptenic molecule.

Various illustrations can be made of the different combinations whichmay be employed in the flow modulation methods of the subject invention.For example, the reagents could include an agarose bead to which isbound polyclonal antibodies to an antigen of interest or monoclonalantibodies where different particles are specific for different epitopicsites of the antigen. The sample need only include the detectioncomponent and any of the analyte which may be present. The sample wouldmix with the antibody-conjugated particles in the reaction unit and thenflow into the exit unit. The presence of the analyte would result incross-linking of the particles with large amorphous particles providinga greater drag on flow smaller particles. As the accretion of particlesincreased, ultimately a plug woud form and flow would stop. In the caseof haptens, the sample could be combined with antibodies to the haptens.The reagent could be hapten conjugated to Porglas beads. Availableantibody would be capable of cross-linking the hapten-conjugated beadsso as to ultimately provide a plug which would inhibit further flow.

Similar techniques could be used with hemaglutination where the particleis a red blood cell to which particular antigens are or have been bound.A sample which might contain cells having as part of their surfacemembrane the same antigen would be combined with the antigen conjugatedred blood cell particles. The reagent would be antibody to the antigen.The rate of formation of the plug would vary depending upon whethercells containing the same antigen were present or absent.

A further illustration would involve polymers to which are attachedpolyclonal antibodies, where the polymers are selected so as to haveonly moderate solubility in the assay medium. Binding to antigenicbridges would result in desolubilization.

The polymers could be conjugated with nucleic acid sequencescomplementary to preselected sequences. The sequences would not becomplementary to the nucleotide sequence of interest or to each other.The sample could be a lysate of a virus or pathogenic organism in anappropriate aqueous medium, where the genomic polynucleotides could besheared. The sample would be combined with the polymeric reagents. Thesample would then react in the reaction chamber with a nucleotidesequence which was a hybrid having a sequence complementary to thenucleic acid sequence of interest and a sequence complementary to thesequence bound to the polymer. Thus, the nucleic acid of interest wouldserve as a bridge to cross-link the polymeric molecules so as to set upa polymeric structure which would substantially slow the flow in thecapillary and could, if desired, provide a plug.

Another exemplification would employ dual receptors, e.g., antibody toanalyte and antibody to a particle, such as an antigen of a red bloodcell (RBC). In quantitating a multivalent antigen, in the presence ofantigen, cross linking would occur between the antigen, dual receptorand RBCs to form large complexes modulating the flow rate, while in theabsence of the antigen no complexes would be formed. The assay could becarried out by combining the sample with RBCs before introducing thesample into the device or by providing RBCs in the sample receivingchamber. The reaction chamber would have the dual receptor where complexformation would be initiated.

For monovalent or haptenic analytes, the assay would be modified byemploying a polyhapten reagent which could be added to the sample priorto introduction of the sample into the device. The presence of haptenwould reduce complex formation in contrast to the result observed withthe multivalent antigen analyte.

Any system which results in a change in flow velocity or can be coupledto a reagent or system which affects flow velocity may be measured.Various systems have already been indicated which result in changes inflow rate. Other systems which could be coupled to compounds of interestare light initiated catalysis of polymerization, cellular aggregationinitiated by lectin cross-linkng, enzymatic reactions resulting inpolymer initiation in conjunction with water-soluble monomers, e.g.,hydroxyalkyl acrylates, etc.

It is evident that the system permits a wide variety of variations whichallows for a variety of protocols and reagents. Thus, any substance ofinterest which allows for flow in a capillary can be detected inaccordance with the subject invention.

The flow in the capillary channel unit can be detected by varioustechniques which allow for detection of fluid flow, e.g., flow sensorsor pressure sensors, or by having a detectable component in the assaymedium, which can be detected visually or by diode assay. Techniqueswhich allow for fluid flow determinations include the use of means formeasuring triboelectricity, means for detecting the rate of passage ofliquid, detecting Doppler effects, or the like. Preferably, a componentis used in the medium which allows for flow detection by detecting thepassage of the component through the first capillary channel exiting areceiving chamber.

Flow can be detected by the creation of a speckle pattern resulting fromthe movement of particles in the first capillary channel and the passageof a coherent light source, e.g., laser beam, or an LED, through thechannel. (See, Powers et al., supra.)

A speckle pattern results from the interaction of particles and coherentlight. Flow (motion) of the particles makes the speckels move with afrequency associated with the flow rate and the light or specklefluctuations can be detected by a photodetector. The photodetector isdesigned to detect an area not greater than about the size of a speckle.A plurality of photodetector elements may be employed for detectingdifferent areas and averaging the signals from each area. The specklepattern can also be used to determine the size of the particles byanalysis of the size of the speckles.

By employing a photodetector, the change in the light pattern as aresult of a change in the rate of flow can be determined by appropriateelectronic means, such as photodiodes or phototransistors, which wouldfeed the electrical signal resulting from the fluctuating light to anappropriate circuit. Particularly easy to distinguish is a flowingliquid from a stationary liquid. Thus, the slowing or stoppage of flowcan be readily detected and the change in rate of flow or the time ofpassage through the first capillary can be determined from the beginningof flow to the stoppage of flow.

One possible problem that can occur in capillary flow devices of theinvention is depletion of blood or another sample from the reservoirprior to the stoppage of flow caused by the detectable event beingmeasured, such as coagulation. When the liquid in the reservoir is drawndown so that essentially no more fluid is present in the reservoir, flowwill stop since capillary forces will then be operating in bothdirections. Accordingly, it is useful to have a means of detecting thisanomolous result in order to avoid a measurement of flow stoppage causedby this event being taken to represent the measurement flow stoppage.

Since the actual device containing the capillary channels and otherchambers is typically a flat cartridge that is inserted into aninstrument which makes the various electronic measurements, detection ofreservoir depletion can be accomplished by embedding various sensorsinto the electronic device that holds the reaction cartridge.

Since the reservoir is generally external to the electronic device sothat blood or another fluid can be applied directly to the reservoir,measurement of depletion of fluid in the reservoir typically takes placein the presence of ambient light and other ambient conditions, variationin which must be accounted for in any measurement technique. Onesuitable measurement technique is to apply modulated light to thereservoir in a region adjacent to the capillary leading to the reactionchambers and other parts of the apparatus. In fluids containingparticles, such as red blood cells in blood, light is scattered in alldirections through the fluid even though the light is appliedperpendicular to the reservoir. Some light will be scattered down theentry capillary, which will then act as a light guide. However, thepresence of particles in the fluid present in the capillary will againresult in scattered light which passes out through the transparent wallsof the light guide (capillary), where it can be measured by aphotodetector. The capillary channel filled with blood can be consideredto be a leaky waveguide for light, because a difference in refractiveindex between the blood (high refractive index) bounded by a lowrefractive index material (capillary channel) will provide lightguidance, while the presence of red blood cells will scatter the lightthrough the walls of the capillary channel, thereby providing theleaking effect. Since light will only be scattered in the presence ofred blood cells or other particles, a detector located in closeproximity to the channel will detect the scattered light. The modulationof the applied light will isolate the detector from ambientinterferences. If the light is modulated at a defined frequency anddetection electronics are sensitive only to that frequency, ambienteffects will be eliminated. The modulation applied to the light can beof any type, such as sinusoidal waves or chopping, as long as themodulation can be both created and detected by electronic or mechanicalmeans. Interferences from ambient light can further be eliminated byusing infrared light, which offers additional advantages (when blood isthe sample) of enhanced scattering and transmission.

This technique for detecting depletion of fluid in the reservoir offersseveral advantages over other techniques. Detection of fluids incapillary channels is normally accomplished by measuring changes inabsorption or transmission of light passed through the channel. However,in certain instances this will not be possible because of the physicalrestrictions on the reservoir and its location in the capillary deviceand the electronic apparatus into which the capillary device isinserted. For example, the size of a finger, if blood is being obtainedfrom a finger stick, will require that the reservoir be separatedsufficiently from the electronic device to allow the finger to be placedonto the reservoir. This will mean that both sides of the capillarydevice adjacent to the reservoir are not in contact with the electronicapparatus since at least one side must be accessible for the finger.

With the method discussed here, there is no need to have both sides ofthe capillary available for transmission and detection of light. Becausea scattering effect is used, the detector can be present either on thesame side of the capillary on which light is applied, on the oppositeside, or in any other physical relation as long as the detector islocated adjacent to the channel.

An additional useful control device is some means for simulating bloodflow through a capillary channel in order to determine whether theelectronic apparatus into which the cartridge is being inserted is fullyoperational. Numerous means of accomplishing this result are available,but one useful technique not believed to be previously used in anysimilar manner is described below.

As described previously, one useful technique for measuring blood flowis to detect the presence of the speckled pattern that results from theinteraction of particles and coherent light. Any technique thatsimulates blood flow when such a detection system is being used willneed to simulate the speckled pattern of light. Since the detector andthe coherent light source are typically located in a close spatialrelationship directly opposite each other so that insertion of thecapillary device will result in light from the coherent light sourcepassing directly through a channel in the device to the detector,simulation of blood flow requires insertion of some device into theelectronic apparatus that can modulate the light beam. While this couldbe accomplished using a second device that could, for example, producemodulated light, a useful technique is to include electronics andmodulating devices directly in the capillary device so that eachcapillary cartridge can be used to determine the operatingcharacteristics of the electronic apparatus containing the coherentlight source and detector immediately prior to actual measurement beingtaken. However, this requires that the speckled pattern generator besuch that it will not then interfere with the actual measurement. Onemeans of accomplishing these results is to include a liquid crystaldisplay-type apparatus at the location where measurement is being made.The liquid crystal material is selected so as to rotate polarized lightthat passes through it, the typical means by which liquid crystalsoperate. Polarizer filters will be present, either in the cartridgeitself or in the electronic apparatus into which the cartridge isinserted that will result in the passage of light through the polarizingfilters when the liquid crystal device is turned off. However, when theliquid crystal device is activated by application of a voltage, lightpassage will be blocked.

Typically, when the liquid crystal device is activated, it rotates thepolarization of the laser beam, thereby reducing the passage of a lightand generating light amplitude fluctuations, which are detected as beingequivalent to the moving speckled pattern generated by passing coherentlight through the thin film of particle-containing fluid that wouldnormally flow down the capillary channel.

A low viscosity liquid crystal material having a high refractive indexchange (thereby enabling rapid fluctuations) is desirable. A typicaldesign uses a crystal oscillator and a chain of binary counters fromwhich the liquid crystal display driver signals are derived as well asthe time base for the measurements to be taken.

In order to further consideration of the invention, a number ofillustrative devices which may be used will now be considered. Asalready indicated, the device will have at least one capillary channelunit, one chamber unit, an entry port, a vent, and a reagent bound tothe surface.

The device will be fabricated from materials with the appropriatephysical properties, such as optical transmission, thermal conductivity,and mechanical properties, and which allow for uniform coating andstability of reagent, as well as medium compatibility, for example,blood compatibility. Where blood is the medium, the material should beconfigured to assure good blood flow stoppage or slowing once clottingis initiated. For this purpose, suitable plastics include those for highsurface free energies and low water sorption, including PETG, polyester(Mylar®), polycarbonate (Lexan®), polyvinyl chloride, polystyrene, andSAN. A particularly preferred plastic is acrylonitrile-butadiene-styrene(ABS), particularly ABS supplied by Borg Warner under the tradenameCycolac. However, since these plastics are hydrophobic and exhibit poorreagent coating and poor blood flow, the plastics can be renderedhydrophilic by treatment with argon plasma, using a plasma etcher orcorona discharge. Suitable conditions are 10-25 watts at 13.56 MHz andone torr chamber pressure for 5-10 min. Alternatively, a protein, e.g.,albumin coating, can be used in some instances by passing a solutionthrough the device having from about 1-5% serum albumin, allowing thesolution to stand for 30 min., wiping and drying. Other modificationsmay also find application. Plasma etching and corona discharge providemarkedly superior flow control characteristics and reproducibility.

The device can be fabricated in a variety of ways. The receiving andreaction chambers can be formed in the plastic sheed by vacuum forming(PETG), injection molding (PETG, polystyrene, SAN), or hot stamping.Capillaries may be formed by etching a channel into the plastic. Thedevice can be sealed by placing a cover slip (with appropriate ventholes at the inlet and vent) on the base sheet, and sealing withultrasonic welding or by solvent bonding. Of these techniques, markedlysuperior products are obtained by injection molding of the plasticdevice in pieces so as to form a depression in at least one surface ofat least one plastic piece. ABS polymers are particularly suited toinjection molding and additionally provide a clear plastic which issuitable for numerous optical detection techniques. ABS polymers arealso suitable for ultrasonic welding. It is preferred to form thechambers from two substantially flat plastic pieces in which thecapillaries and other chambers are formed by producing matchingdepressions in two surfaces of two different shaped plastic pieces. Itis preferred that on one of the pieces ridges, known as energydirectors, completely surround the depression in a closely spacedrelation so as to form a surface of first contact when the two piecesare placed together. When ABS is used, the ridges are typically 7.5 mil±0.5 mil above the surface of the plastic. The ridges are typicallyformed in the shape of a triangle, typically an equilateral triangle.The center of the ridge is typically 17.5± 0.5 mils from the edge of thedepression that will form the chamber. Use of such energy directors withultrasonic welding produces a highly reproducible seal around the edgesof the internal chamber that is formed when the two sheets areultrasonically welded together. Access ports are typically formed bymolding or drilling holes into the depressed surfaces of the individualplastic pieces prior to welding. Accordingly, the welded ridges form acomplete seal around the lateral edges of the internal chambers.

Alternatively, the pattern can be dye cut in a double-sided adhesivetape (e.g., 3M No. 666 tape, Fasson Fastape A) of appropriate thicknesswhich is then sandwiched between a plastic base and cover slide. Or, thesandwiched layer may be die cut from a plastic piece of appropriatethickness which would be coated with adhesive and sandwiched in the samemanner as the tape. The adhesive could also be silk-screened onto thebase to give a raised pattern of desired thickness.

The sheet thickness of the device in the region of the capillarychannels will generally be equal to or exceed about 2 mil to preventcompression due to the capillary action. In the embodiment involving thesandwich, each of the plastic layers comprising the top and bottom willbe at least about 10 mil thick.

While other materials may be used for fabrication, such as glass, forthe most part these materials lack one or more of the desirablecharacteristics of the indicated materials and therefore have not beendiscussed. However, there may be particular situations where glass,ceramic or other material may find application, such as a glass windowfor optical clarity, modification of surface tension, and the like.

The device will normally include a reagent within the reaction chamber.In formulating the reagent(s), it may be formulated neat or with variousadditives. The manner in which it is formulated, introduced into thereaction chamber and maintained in the reaction chamber, must providefor rapid mixing with the sample, reproducible distribution in thechamber, stability during storage, and reproducible reaction with thesample.

In order to assure the reproducibility of distribution, varioustechniques may be employed for introducing the reagent into the chamber.Where the device is produced as two parts which fit together, thereagent may be sprayed, painted, introduced into the chamber as aliquid, lyophilized or evaporated, adsorbed, covalently conjugated, orthe like. The active reagent may be combined with various stabilizers,excipients, buffers or other additives involved with the reaction.Alternatively, a small vial or other holder may be attached to thereaction unit, usually chamber, being stored as a liquid, where theliquid may be introduced into the reaction unit prior or concurrentlywith the sample entry into the reaction unit. A second receiving chambermay be employed connected to the reaction unit by a capillary channel,where transfer of the reagent in the second receiving chamber to thereaction unit is initiated in relation to introduction of the sample.For example, the second receiving chamber could be filled and sealed,and then unsealed when the sample is introduced into the samplereceiving unit.

To enhance mixing, various mechanical or ultrasonic means may beemployed to agitate the sample and reagents, where the mixing means maybe internal or external. Vibrators, ultrasonic transducers, magneticrods or other mechanical mixing means, flow disrupters, mixing bafflesor barriers, flow directors, or the like, may be employed. Theparticular manner in which agitation is provided, if provided, will varywidely depending upon the degree of agitation needed, the design of thedevice, and the like.

Various chemicals can be used to ehance dissolution in a uniform manner.Such chemicals may include surfactants, polyols, sugars, emollients,liquids, or the like. Depending upon the nature of the reagents, thereagents may be formulated in a variety of ways to insure rapid anduniform mixing.

Other chemicals can also be present in the reagent chambers. Forexample, if the device is being used to measure prothrombin time and acontrol sample containing heparin is being used, such as described in anapplication filed on even date with the present application and entitled"Whole Blood Control Sample", which is herein incorporated by reference,said application being assigned to the same assignee as the presentapplication, a heparin antagonist can be used to eliminate the effectsof heparin on prothrombin time measurement. Typical heparin antagonistsinclude protamine sulfate and polybrene.

The reagent need not be coated or bound to the surface of the device,but may be provided as a soluble sponge or gel or alternatively,absorbed onto an insoluble sponge, membrane, paper (e.g., filter paper)or gel which is introduced into the reaction unit. In this manner thefluid may pass through the foam structure dissolving the reagent so asto form the reaction mixture.

The reagent may be provided in liquid form in microcapsules. The liquidreagent could be released from the microcapsules by applying pressure tothe walls of the reaction unit, resulting in breaking of themicrocapsules and releasing the liquid reagent.

Also, as already indicated, the reagent need not be limited to a singlereaction unit. The same or different reagents may be introduced into thecapillary or in successive reaction units. In this manner a cascadingreaction may be performed, where one is interested in allowing eachreaction step of a sequence to proceed for a predetermined period beforeencountering the next reagent. Multiple reaction units also allow forthe removal of components in the sample which may interfere with thedesired reaction. By having receptors in the first units, one or moreendogenous components may be removed. Where particles are to be removed,filters may be employed at the entrance or exit to a reaction unit.

In addition to the chemical reagent, microparticles may be also includedin the reaction unit which would be entrained with the moving front,where the microparticles could aid in the plug-forming mechanism forflow stoppage.

In performing the assay, a sample would be taken and treated as may beappropriate. Blood for example might be diluted and various reagentsadded, particularly where there is an interest in the determination of aparticular clotting or anit-clotting factor. In specific binding assays,various particles might be added which had been functionalized by theaddition of specific binding members, such as haptens, ligands, andreceptors, particularly antibodies. In some instances, the system may bedevised where clotting will occur in the absence of the analyte. Thus,reagents will be added which, in the absence of the analyte, would bedegraded in the reaction chamber.

Once the various materials are mixed to form the sample medium, thesample medium would be introduced into the receiving unit andtransferred by capillary action into the next unit. Either visualevaluation of the flow rate change or an electro-mechanical evaluationmay be employed. The initiation of flow through the first capillarychannel or through a successive capillary channel may be selected as theinitiation time for measurement, or some point in between. As alreadyindicated, various means may be employed for determining the flowvelocity or time to flow stoppage.

For measuring a speckle pattern, which is obtained with particles, asare present in blood, an apparatus comprising a semiconductor laser andphotodectors may be employed. By exposing a photodetector ofsufficiently small area to a speckle pattern, a random signal (noise) isobserved. The average of the random signal observed as a DC signal isinversely proportional to the red cell density, and changes in thefluctuation continues until flow stoppage, e.g., clotting, occurs. Suchapparatus may include a housing for receiving and holding the device andmeans for controlling the temperature.

The size of the area which is detected by an individual photodetectormay be controlled in a variety of ways. One way, as indicated above, isto use a photodetector which has only a small photosensitive area, up toabout the size of the speckle spot. Another way is to use an opticalfiber. By controlling the parameters of the fiber, the area from whichthe fiber receives light may be controlled. Instead of a fiber, lensesmay be employed to limit the observed area which lenses may be separatefrom or molded into the device.

Where other than flow stoppage is involved, various spectrophotomers,fluorimeters, or the like, may be employed for detection of thedetectable signal. Depending upon the nature of the assay protocol, asingle determination or multiple determinations may be made, based on afixed value or a kinetic determination.

Various devices may be devised for the subject assays. In FIGS. 1A and1B devices are depicted involving single chambers and one or twocapillary units. These devices can be fabricated in a variety of ways,for example, having two sheets, where each of the sheets have beenmolded so as to define the particular units or one of the sheets definesthe units and the other is a cover sheet, or having three sheets, wherea sheet having cutouts defining the units is sandwiched between theother two sheets, where one or the other sheets provides the necessaryorifices for the various ports. Other techniques may also be found to beuseful for providing the chamber and channel cavities.

In employing device 10 of FIG. 1A, capillary 12 is introduced into thesample, so that the inlet port 14 is completely submerged in the sample.It is important to avoid any air bubbles where the air bubbles couldinterfere with the measurement. The inner surface of the upper portionof capillary 12 is coated with reagent 16, so that as the liquid sampletransits the capillary 12, the reagent 16 becomes dissolved in thesample. When the liquid front reaches index 18, the capillary is removedfrom the sample, defining the sample volume, and the capillary insertedinto a second fluid to maintain continuous flow. Otherwise, the sampledrop can maintain a reservoir outside of the inlet port 14. The sampleflows through capillary 12 into chamber 20 having vent 22. A secondreagent 24 is coated onto the inner surface of reaction chamber 20,where the assay medium undergoes a second reaction.

In using this device, an assay medium could be prepared as the sampleinvolving the fluid suspected of containing the analyte and a bufferedmixture of enzyme-analyte conjugate. The reagent 16 would be antibody tothe analyte, so that the enzyme-analyte conjugate present in the assaymedium would become bound to the antibody in an amount related to theamount of analyte in the assay medium. The assay medium would then enterthe chamber 20, where the reagent would be substrate for the enzyme. Onecan employ a substrate of limited solubility, so that the amount ofsubstrate rapidly reaches equilibrium and remains constant during themeasurment. One can also have a high concentration of a solublesubstrate to maintain substrate concentration constant during themeasurement. One can then determine the rate of formation of productwhich will be dependent upon the amount of active enzyme present in thechamber. Since the amount of active enzyme can be related to the amountof analyte, this rate will therefore be proportional to the amount ofanalyte in the sample. By employing a substrate and enzyme whichproduces a colored or fluorescent product, the rate can be monitored bythe change in color or change in fluorescence over a predetermined timeperiod.

In FIG. 1B, device 30 has capillary 32 which is divided into channels 34and 36 containing reagents 38 and 40, respectively. The two channelsshare a common inlet port 42. Channel 34 contains reagent 38, referredto as the first channel and the first reagent, which could bemicroparticles to which are conjugated antibodies to a first epitope.Channel 36 and reagent 40, referred to as the second channel and thesecond reagent, would contain microparticles having monoclonalantibodies to a second epitope. In each case, the amount of monoclonalantibody would be substantial excess of any analyte which would beencountered. The analyte of interest would have a single epitope bindingto the first reagent and a single epitope binding to the second reagent.The sample would travel through the channels at a substantially constantrate, with reaction occurring with any substance having the appropriateepitope.

All of the components present in the assay medium having the appropriateepitopes would react with the particle conjugates and become bound tothe particle conjugates. The particle conjugates would then exitchannels 34 and 36 and enter incubation chamber 44. The chamber wouldalso provide for capillary action, and agitation due to its accordianshape and vanes 46 for causing turbulence in the chamber 44. Thus, asthe assay medium exited the channels 34 and 36, the microparticles wouldmix and cross link, if any analyte was present which had the twoepitopes on the same molecule.

In the incubation chamber, the particles would have sufficient time toaggregate, so that upon entry into exit capillary 48, the particleswhich are formed would have a significant effect on the flow rate incapillary 48. By measuring the rate of flow or determining particle sizeor number of particles in capillary 48, one could determine the presenceand amount of an analyte having both epitopes. The rate of flow or otherparameter could be determined by the rate at which particles above acertain size transited a light path, using minimum light intensityfluctuations, level of scatter, or the like.

The subject device illustrates the opportunity for having a plurality ofcapillaries to divide a sample into a plurality or portions, where eachof the portions can be treated differently. The differently treatedportions may then be brought together into a single chamber, where thedifferent portions may interact in accordance with the desired protocol.Depending upon the nature of the protocol, the resulting assay mediummay then be transferred to a capillary which may provide for measurementor may be further transferred to additional chambers for furthermodification.

In FIGS. 2A and 2B are depicted a device having a plurality of chambersand allowing for interrupted flow. The device 50 is fabricated fromthree sheets, an upper sheet 52, a lower sheet 54, and a spacing sheet56, which defines the various capillary units and chamber units. Thedevice has three chambers, the receiving chamber 58, the reactionchamber 60, and the effluent chamber 62. Inlet port 64 receives thesample which is measured by filling chamber 58 and reaching the firstcapillary 66. Receiving chamber 58 is coated with first reagent 68, sothat the sample undergoes a reaction in receiving chamber 58 and ismodified.

The modified sample then passes through capillary 66 and enters reactionchamber 60, which is coated with second reagent 70. The second reagent,like the first reagent, is part of a detection system to provide for adetectable signal. Vent 72 is provided to stop the flow and allow forincubation in reaction chamber 60. This can be particularly useful wherethe slow step in the development of the detection system is complexformation. Depending upon the nature of the protocol, the period ofincubation may be specifically timed incubation or may be one which isallowed to sit for a sufficient time to ensure completion and then thedetermination made. Effluent chamber 62 has exit vent 74 to permit flowpast intermediate vent 72 when intermediate vent 72 is closed. Uponclosing of intermediate vent 72, the assay medium will then flow throughcapillary 76 into effluent chamber 62. Instead of sealing the vent,other alternatives, such as applying pressure or centrifugal force, maybe used to restart flow.

The device is comprised of a block 78 which may be configured to beintroduced into a instrument for assay determination. As alreadyindicated, the various sheets will be constructed so as to ensuresufficient mechanical stability to withstand capillary action andprovide for the necessary characteristics for flow of the assay mediumand detection of the detectable signal.

The subject device may be employed for flow stoppage, such ascoagulation, where the coagulation may occur in the effluent chamber 62.One could measure the rate of flow in capillary 66 or determine the timeof flow stoppage, particularly where capillary 76 is elongated (See FIG.5) and a plug forms in capillary 76.

In this device, one could provide for a determination of particle count,where the first chamber has bead conjugated ligands and the analyte ofinterest is a particular antibody. The sample would be introduced intothe receiving chamber 58, where reaction would occur between the beadconjugated ligands and any antibody present in the sample. The samplewould then flow to the reaction chamber 60, which would contain areagent which binds specifically to antibody-ligand complexes, such asS. aureus protein A or rheumatoid factor, which binds specifically topoly(antibody-ligand) complexes. Thus, any bead conjugate which becomesbound to antibody, would be removed from the liquid phase of the assaymedium. The device 50 could then be inserted into an instrument, whichwould cover vent 72, allowing for flow through capillary 76. One couldthen determine the number of particles or beads in the assay medium inthe chamber 62 or capillary 76 as a measure of the amount of antibody inthe sample.

Alternatively, one could provide for complex formation between Fabfragments and major histocompatibility antigens of cells in thereceiving chamber 58. Thus, reagent 68 would be Fab fragments ofmonoclonal antibodies specific for the major histocompatibility antigen.The Fab fragments could be from murine or other non-human source ofmonoclonal antibodies. The reagent 70 would then be particles to whichanti-murine immunoglobulin were conjugated. In this way, when theFab-bound cells entered the reaction chamber 60, they would bind to thelatex particle conjugates so as to form extended structures. Uponclosing of the vent 72 by introducing device 50 into an instrument, themedium would flow through capillary 76, where large particles could bedetermined by the scattering of light, or the pattern of transmission oflight through the capillary and blockage by the cellular-particleaggregations.

In FIG. 3 is depicted a device which is exemplary of the determinationof a plurality of anlaytes in a single sample. The device 80 has areceiving chamber 82 with inlet port 84.

The sample would be introduced into the receiving chamber 82 and bepumped by capillary action through channel 86 into reaction chamber 88.In reaction chamber 88 would be one or more reagents 108 which wouldprovide part of the detection system. From reaction chamber 88, theassay medium would then be pumped by means of capillaries 90, 92 and 94to chambers 96, 98 and 100, respectively. The media in chambers 96 and98 would then be pumped by means of side capillaries 102 and 104 tofinal chamber 106. In this way, a variety of reactions could occur,where reagents could be provided in the various side chambers forfurther reaction allowing for detection of a plurality of epitopicsites.

An illustrative of the above apparatus, one could determine from alysate, the serotype of a particular pathogen. The lysate would beintroduced through inlet port 84 into the receiving chamber 82 and thenbe pumped into the reaction chamber 88. In the reaction chamber would bereagent 108 which would be monoclonal antibody-bead conjugates to apublic epitope of the particular pathogen. One would then measure theparticle count in capillaries 90, 92 and 94, which would provide thebase line for the particle count which should be present in chamber 96,98 and 100. In each of the chambers 96, 98 and 100, would be monoclonalantibodies to an epitope specific for a particular serotype, where theantibodies are conjugated to larger beads which are distinguishable bylight scattering properties from the beads in chamber 108. Thus, if thesignal character changes in chambers 96, 98 or 100, this would beindicative of the particular serotype.

If one wished to determine if the serotype had another antigen ofinterest, one could provide for antibodies to the particular antigen inchamber 106. Chamber 106, a narrow chamber susceptible to particleblockage, would have vent 110 to allow for flow into chamber 106.Capillaries 102 and 104 would pump the assay medium from chambers 96 and98 into chamber 106, where the presence of the particular antigen wouldresult in cross linking of the antigen. Cross-linking of the antigenwould result in plug formation.

Other combinations of labels and protocols may be employed. By dividingthe assay medium into multiple pathways, the assay medium can be treatedin multiple different ways and, if desired, rejoined in a single chamberas indicated above. This can be useful in situations where one isinterested in different analytes, which may have different combinationsof epitopes, or where the analyte must be treated in different ways inorder to provide the detectable signal, or where one wished to adddifferent combinations of labels to the analyte, where one wishes toprovide a check on the results observed, or the like.

In FIG. 4 is depicted a device which allows for the simultaneousdetermination of a sample background value and the detectable signal.The device 120 has an inlet port 122 separated by partition 124 whichextends through capillary 126, chamber 128 and second capillary 130.Capillary 130 evacuates into effluent chamber 132 having vent 134.Chamber 128 is divided into two half chambers or semichambers 136 and138. In semichamber 136, two reagents are present indicated by theslanted lines and the crosses. The slanted lines are monoclonalantibodies specific for an epitope on the analyte, where the antibodiesare non-diffusively bound to the surface. The crosses indicatemonoclonal antibodies conjugated to fluorescers, where the monocolonalantibodies bind to a different epitope of the analyte. The fluorescerconjugate is reversibly bound to the surface of the two chambers 136 and138 in the area near the entry ports 140 and 142 of capillary 126.

In carrying out the assay, the sample inlet port is submerged into thesample and the sample allowed to rise in the capillary 126. Sufficientsample is introduced and the two chambers 136 and 138 are filled.

As the sample transits the two semichambers, different events willoccur. In the sample reaction chamber any analyte will become bound bothto thee antibody bound to the surface and the fluorescer conjugate, sothat the amount of fluorescer conjugate which remains in the samplereaction chamber 136 will be dependent upon the amount of analyte in thesample. By contrast, sample which traverses control reaction chamber 138will bind to the fluorescer conjugate but continue through the chamberinto capillary 130.

By appropriate optics, one can read the fluorescence from the twocapillary regions 144 and 146. The capillary region 144 will be theregion for determination of the amount of analyte, while the capillaryregion 146 will serve as the control. Thus, the amount of fluorescenceobserved in region 146 will be the maximum amount of fluorescenceavailable from the combination of sample and fluorescer conjugate. Anyreduction in fluorescence in the capillary region 144 will be as adirect result of the presence of analyte. The two streams will then exitinto effluent chamber 132.

As shown in FIG. 5, the next embodiment 160 provides a serpentine path.The device has a housing 162 which is a rectangular plastic block shapedto fit into a reading apparatus (not shown). The block is indexed atsite 164 for alignment to the apparatus. The receiving chamber 166 has avolume about one and one-half times the volume of the reaction chamber.The two chambers are connected by the first capillary channel 168. Inletport 170 provides for introduction of the assay sample by syringe,eyedropper, or other convenient means. A serpentine capillary path 172connects to outlet 174 of reaction chamber 176. The serpentine channel172 terminates in reservoir chamber 178 which has outlet port 180.

Various other configurations can be employed in particular situations.For example, one could employ a "Y"-shaped device where one arm of the Yhas a sample receiving chamber with the inlet port sealed. The samplereceiving chamber is connected through a first conduit to the mixingmember which serves as the trunk of the Y. The first conduit may befilled with a fluid which may be a buffer solution or other diluent.

The second are of the Y has a fluid chamber with a removalbe seal over aport. The fluid chamber is filled with a fluid which may serve as adiluent, reagent source, or the like. The fluid chamber may be as largeas desired so that it may provide the desired ratio of fluid to sample.The fluid chamber is connected to the mixing member through a secondconduit which is also filled with the fluid of the fluid chamber. Thecross-sections of the first and second conduits are selected to providefor the proper volume ratio of the fluid and sample.

The mixing member may be a capillary or chamber. The mixing member maybe the final element of the device or may be connected to additionalelements. Thus, the Y can serve as a complete device, by providing themixing member with an outlet port or a part of a larger device byconnecting the mixing member to additional capillaries and/or chambers.

To use the Y, the seals are removed from the fluid chamber and samplechamber, while retaining the outlet port sealed to prevent flow. Theinlet port may then be contacted with the sample and the outlet portunsealed. The sample and fluid from the two chambers will begin to flowand be mixed in the mixing member in the proper proportion. Depending onthe particular protocol, one can determine a number of different eventsby reading the flow of fluid in one of the capillaries.

One application would be to provide fluorescent antibodies in the fluidchamber. Where the sample contains cells having the homologous ligandthe mixing of the fluorescent antibodies and cells will result in largefluorescent particles as a result of the homologous antigen beingpresent on the cells. These large fluorescent particles could then bedetected by various means.

FIG. 6 shows a cross-sectional view through a section of a hypotheticaldevice being prepared by ultrasonic welding from two injection-moldedplastic pieces (not necessarily shown in any of the previous figures),prior to ultrasonic welding. The two plastic pieces 202 and 204 thatwill be used to form the internal chambers are shown properly aligned(i.e., in register) in FIG. 6A. "Registration" is used here in theprinting sense, referring to proper alignment of the depressions presentin the surfaces of the two pieces that are used to form the internalchambers and capillaries. Proper registration can be aided by injectionmolding the two pieces to provide projections on one piece that fit intoholes or depressions (other than capillary- or chamber-formingdepressions) in the second piece. A single convoluted depression, 206and 208, respectively, is formed into the surface of each piece, but thecross-sectional view shown in the figure cuts through the depression atthree separate locations, two of which will result in capillary spaces(210 in FIG. 6B) while the remaining location will result in theformation of a larger reaction chamber (212 in FIG. 6B).Energy-directing ridges 214 can be seen in the surface of one of the twoplastic pieces (204) adjacent to the periphery of the depression.

FIG. 6B shows the same cross-sectional view after ultrasonic welding.The plastic has melted selectively in the region of the energy-directingridges so that the two plastic pieces have melted into each other toform a seal around the capillary and the reaction chamber. In order tominimize the destructive effects of heat caused by the ultrasonicwelding, ultrasonic welding is carried out only until a seal is formedand does not need to be carried out until the entire plastic surfaceshave welded together. Unwelded contact surfaces are shown by referencenumbers 216 in FIG. 6B. Use of energy ridges and short welding timesalso ensure that the dimensions of the depressions will be unaffected bythe welding event. Welding time will be selected so that the melting(welding) almost but not quite reaches the edge of the depression. Theextremely small cracks left between the two plates in the area of thecapillaries will not adversely effect capillary action.

FIG. 7 shows electronic circuitry that can be utilized to simulate thepassage of blood through a capillary flow device. The circuitry includesa crystal-controlled oscillator in which 220 represents the crystal and222 represents the oscillator. The signal from the oscillator drives twofrequency dividers (224 and 226) that will generate the output signalsfor a driver 228 of a liquid crystal display cell 230. Cell 230 isbiased by an oscillating signal having a specific rate of oscillation,for example 128 Hz. The cell will therefore rotate its polarization atthe rate of 128 Hz. Polarizer 231 in combination with cell 230 thereforeoperate to alternately block and pass light as a result of the rotatingpolarization.

Two more dividers (232 and 234) drive a logical AND gate (236) whoseoutput will go to a logic circuit low at defined intervals, for example,approximately every 20 seconds. When the output goes to a logical low,the output of a logical OR gate (238) will reset the dividers, therebystopping the process. Accordingly, modulating signals for the liquidcrystal display cell 230 are generated for the set time period, 20seconds in the above example.

The device is provided with a start switch 240. When switch 240 isclosed, the reset signal is cleared, and the process is restarted.

The oscillator, dividers, logic gates, and liquid crystal display drivercan be implemented in CMOS technology using standard techniques ofelectronic fabrication. An CMOS device can be readily powered throughmore than 10,000 cycles when powered by a coin-type lithium battery.

FIG. 8 shows a device for determining when the sample present in areservoir is depleted. In this figure, blood is presumed to be thesample. When blood is applied to sample reservoir 242 it will startflowing immediately down capillary channel 254 by capillary action. Alight source 246, typically an infrared light-emitting-diode (LED), islocated adjacent to the blood reservoir near the capillary entrance.Light source 246 is modulated by a modulator 248, typically a sinusoidalwave or square wave generator. A typical modulation frequency is about 8KHz. The modulated light will be scattered by red blood cells in theblood sample, and a fraction of this light will be guided by capillarychannel 244 formed in the plastic capillary flow device 250. Since thered blood cells in the sample will further scatter this guided light,photodetector 252, typically located in close proximity to the inlet 254of the capillary channel, will capture some of the scattered light. Thesignal output of the photodetector will consist of the superposition ofambient light and scattered light. The scattered light component isseparated from the ambient light by a band-pass filter 256 and isfurther amplified by amplifer 258. This signal is rectified by rectifier260 and integrated by integrater 262 in order to generate a directcurrent voltage proportional to the scattered light. Other types ofsignal generators can be used to produce a detectable signal that isseperable from ambient light and its possible variations. Examplesinclude wave lengths (e.g., use of infrared light sources), lightpulses, sinusoidal wave generation, and digital encodation. When methodsother than frequency modulation are used to produce the detectablesignal, the term "filter" as used in this specification refers to anymeans of separating the detectable signal from variation in ambientlight.

Although the exact location of the ambient light source and detector inrelation to the junction between the blood reservoir and the capillarycan be varied depending on the capillary size, strength of a lightsource, detection limit of the detector, adsorbance of the sample, andthe like, it is preferred that both the light source and the detector byrelatively close to the junction, particularly when a highly abosrbantsample such as blood, it is utilized. When an infrared light source isused, it is preferred to place the light source from 0.5 to 2 mm fromthe capillary entrance with an infrared-sensitive photodetector beinglocated from 1 to 4 mm from the junction.

As the blood reservoir empties due to the capillary flow, the light pathbetween light source 246 and photodetector 252 will be interrupted,thereby reducing the voltage output from integrator 262. By connectingthe output of the integration to a comparator 264, a logical levelindicating the presence or absence of sample in the reservoir isobtained. Furthermore, by adjusting the position of the light source orthe reference voltage of the comparator, the volume of sample in thereservoir at which the decision "no sample" is made can be controlled.

An infrared light source is preferred because of its larger efficiencyin converting electric current into light, as compared to visible-lightlight-emitting-diodes. A modulating frequency of several kilohertz(preferably 3 to 20 kHz) is selected in order the move the modulationfrequency from the low 60 Hz harmonics present in artificialillumination, thereby simplifying the separation of ambient light andsignal light. The separation is enhanced even further by the selectionof an infrared light source.

Obviously, various designs of the individual chambers and channels canbe provided. The designs and channels will be selected to provide foroptimum sensitivity for particular assays. The volumes of the chamberswill be chosen so as to accommodate an appropriate sample volume. Thenature and cross section of the first capillary channel together withthe size of the reaction chamber will control the residence time of theassay medium in the reaction chamber. In some systems a reaction willterminate upon the sample exiting the reaction chamber, e.g.,antigen-antibody complex formation, pro-enzyme to enzyme, etc., where acomponent is bound to the surface of the chamber. The reaction occurringin the reaction chamber, may result in a product which produces ablockage in the second capillarly channel or prevents a blockage fromforming. The residence time for the reaction in the reaction chamber canbe carefully controlled by controlling the dimensions of the capillarychannels and reaction chamber, as well as temperature.

It is evident that any type of capillary channel may be employed whichprovides for accommodating the appropriate volume and time period toflow stoppage. Various designs may be used such as serpentine, linear,U-shaped, pleated, or the like. The channel cross-section may becircular, ellipsoid, rectangular, or combinations thereof. The length ofthe channels may be determined empirically depending upon the otherparameters involved.

The initial or metering channel may be of constant or varyingcross-section. With a constant cross-section, the observed flow velocitywill diminish with the path length traversed. Therefore, the observedchange in velocity will have two components: (1) an inherent reductionin velocity related to the increasing friction with increasing fluidpath length; and (2) increasing or decreasing viscosity of the mediumdue to any reaction occurring.

In order to eliminate the effect of the fluid path length, a taperedcapillary may be employed. The taper can be calculated by determiningthe cross-section, e.g., height and width, of the channel for each pointalong the channel path. The equations below are employed. The equationsare based on known principles of fluid mechanics (e.g., R. Byron Bird,Warren E. Stewart, Edwin N. Lightfoot, Transport Phenomena, John Wiley &Sons, Inc., 1960).

The flowrate, Q, measured by the laser (in the first capillary channel)can be defined by: ##EQU1## where, V is the velocity in the 2ndcapillary, and A is the area of the 1st capillary channel, and:

r=radius of the 2nd capillary channel

μ=viscosity of the fluid

z=distance down the 2nd capillary channel

ΔP=the pressure drop in the 2nd capillary

is defined by: ##EQU2## wherein: γ=surface tension of the fluid

φ=contact angle of fluid with surface

Combining (1) and (2), the flowrate, Q, becomes: ##EQU3## Form (3) itcan be seen that the radius of the 2nd capillary channel is proportionalto the distance down the channel, z: ##EQU4## and Q* is the desiredconstant flowrate in the 2nd capillary channel. Using the aboveequations and selecting a desired Q*, the following table indicates thechanges in radius at the position defined by z, for k=0.034

    ______________________________________                                        Length                                                                              Radius   Flowrate Inc Vol Vol   Flow Time                               mm    mm       mm.sup.3 /sec                                                                          mm.sup.3                                                                              mm.sup.3                                                                            sec                                     ______________________________________                                         5    0.090    0.206    0.13    0.127 0.62                                    10    0.090    0.103    0.13    0.254 1.85                                    15    0.090    0.069    0.13    0.382 3.70                                    20    0.092    0.056    0.13    0.515 6.11                                    25    0.099    0.056    0.16    0.671 8.91                                    30    0.106    0.056    0.18    0.846 12.06                                   35    0.111    0.056    0.19    1.040 15.56                                   40    0.116    0.056    0.21    1.253 19.38                                   45    0.121    0.056    0.23    1.482 23.51                                   50    0.125    0.056    0.25    1.729 27.95                                   55    0.129    0.056    0.26    1.992 32.68                                   60    0.133    0.056    0.28    2.270 37.68                                   65    0.137    0.056    0.29    2.563 42.97                                   70    0.140    0.056    0.31    2.872 48.52                                   75    0.143    0.056    0.32    3.195 54.33                                   80    0.147    0.056    0.34    3.532 60.40                                   85    0.149    0.056    0.35    3.883 66.72                                   90    0.152    0.056    0.36    4.248 73.28                                   95    0.155    0.056    0.38    4.626 80.08                                   100   0.158    0.056    0.39    5.017 87.12                                   105   0.160    0.056    0.40    5.421 94.40                                   110   0.163    0.056    0.42    6.838 101.90                                  115   0.165    0.056    0.43    6.267 109.63                                  120   0.168    0.056    0.44    6.709 117.58                                  125   0.170    0.056    0.45    7.163 125.75                                  130   0.172    0.056    0.47    7.629 134.13                                  135   0.174    0.056    0.48    8.107 142.73                                  140   0.177    0.056    0.49    8.596 151.55                                  145   0.179    0.056    0.50    9.098 160.56                                  150   0.181    0.056    0.51    9.610 169.79                                  155   0.183    0.056    0.52    10.134                                                                              179.22                                  160   0.185    0.056    0.54    10.669                                                                              188.85                                  ______________________________________                                    

It is evident from the above table that the path may change from acapillary of constant radius, to one in which the radius increases(e.g., a funnel) or decreases with distance. This formula can be used tovary velocity as desired as the liquid moves down the capillary track orthrough a region of reagent. On could even envision a pulsating flow.

In accordance with the subject invention, novel devices and methods areprovided for measuring a wide variety of sample characteristicsincluding analytes of interest. The devices provide for simplemeasurement or volumes, mixing of reagents, incubations, and visual orinstrumental determination of the result. The detection system mayinvolve the absorption or emission of light, or modulation of flow,including slowing, stoppage, or their reversal. Of particular interestis the use of blood where clotting can be determined or reagentsaffecting clotting. Also of interest are a wide variety of analytes,which include naturally-occurring compounds or synthetic drugs. Thedevices allow for the simultaneous performance of controls andcomparison of signals from the two media. In addition, variouscombinations of channels and chambers may be employed, so that thepathways can diverge and converge, be broken up into a plurality ofdifferent pathways or a sample may be divided into a plurality of pathsand treated in a variety of ways. The devices can be simple to fabricateand the serpentine paths readily devised by employing known fabricationtechniques, with particularly advantageous devices being availablethrough use of the preferred fabrication techniques described herein.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1 Detection of Prothrombin Time

A device or cartridge analogous to the device of FIG. 5 is employed. Twopieces of cellulose acetate 40 mil thick are separated by scored Fasonfast tape B to provide the proper design of channels and chambers. Thereaction chamber contains a thromboplastin reagent where an aqueoussolution of the thromboplastin is introduced into the chamber, the waterevaporated, leaving the chamber coated with thromboplastin. Thethromboplastin reagent composition is: 15 mg/ml rabbit brainthromboplastin extract; 1% glycine; 0.01% thimerosol; 0.01%streptomycin-sulfate; 0.01% triton X100; 0.08% phenol; 1% polyethyleneglycol 3500; 4% sucrose; 0.001% polybrene™. The mixture is lyophilizedand reconstituted to 0.25 original volume with deionized water. Three μlof the reconstituted liquid is then placed on the reagent area of thedevice and then allowed to air dry before attaching the other sheet orcover of the device. A cover is then placed over the chambers andchannels, and a 15 μl blood sample is introduced into the receivingchamber. The device is then inserted into a monitor which may bethermostatted at 25° C. or 37° C., where a gallium arsenidesemiconductor laser of wavelength 0.78μ directs a beam at a site betweenthe ends of the channel joining the receiving chamber and the reactionchamber. On the opposite side from the laser is a silicon photodetectorof a small area sufficient to detect the oscillating speckled patternresulting from the red blood cells flowing through the channel. A DCsignal is observed with large fluctuations. The DC signal is inverselyproportional to the red cell density, and fluctuation continues untilclotting occurs. The time is then related to known standards todetermine the clotting characteristic of the blood. Where warfarin isbeing administered to the blood source, the time for clotting can berelated to the effect of warfarin on the blood clotting time. Inaddition, the light absorbance in the channel can be determined toprovide for a hematocrit, as a further characteristic of the bloodsample. Because of the short path length of the light, undiluted bloodcan be used which provides a further convenience.

EXAMPLE 2 Detection of Cross-linked Fibrin Dimer by Latex Aggultination

Materials: "Dimertest latex" reagents and control serum (MABCO Ltd). Acartridge analogous to that of FIG. 5 is used. Two flat pieces ofpolystyrene formed by injection moulding are welded together to form thecapillary channel and reagent chamber. The receiving chamber (166) is0.467 cm wide by 0.09 cm deep. The first capillary channel (168) is 1.3mm wide by 0.09 mm deep. The reagent chamber (176) is a elipse of majoraxis 12 mm and minor axis 6 mm and is 0.09 mm deep. The serpentinecapillary path (172) is 160 mm long tapering from a radius of 0.09 mm atthe outlet of the reagent chamber to 0.185 mm at the outlet port (180).

Experimental: Sample (10 μl of either buffer or positive control) wasmixed with antibody coated latex (40 μl); after 3' gentle shaking 40 μlof the mixture was injected into the cartridge containing no reagent andinserted into a monitor as described in Example 1. The signal from thelaser detector was recorded on an Omega chart recorder (model 1202)using 5 V full scale sensitivity and a chart speed of 6 cm/min. Themovement of latex particles through the light beam produced a slightlynoisy trace (.sup.˜ 1 chart division {0.01 full scale} peak-peak).Agglutination of the latex caused by the presence of the analyte(cross-linked fibrin degradation products) resulted in a significantincrease in noise (.sup.˜ 3 chart divisions peak-peak).

EXAMPLE 3 Direct Blood Grouping by Red Blood Cell Agglutination

Materials: Human blood samples of known groups anticoagulated withsodium citrate. Blood grouping antisera (American Dade).

Experimental: Resuspended blood (40 μl) was mixed with antiserum (40 μl)and 40 μl of the mixture injected into empty cartridges (as in example2) and results analyzed as in Example 2. Positive agglutination wasobserved as a rapidly increasing noise level, negative reactions gave asteady, low, noise level.

EXAMPLE 4 Direct blood grouping by flow stop

Materials: Blood samples as in Example 3. Cartridges as described inExample 2 were employed. Before welding the two parts of the cartridge,the serpentine track of the lower part was evenly coated with 5 μl bloodtyping anti-serum (American Dade) which had been dialysed against 1%glycine (Na+) pH 7.5 containing 0.01% Triton X-100, 2% sucrose and 0.5%polyethylene glycol -3500. Solvent (water) was then removed byevaporation and the two parts of the cartridge welded together.

Experimental: Blood (40 μl) was injected into the cartridges afterinsertion in a monitor (described in example 1). The time for flow ofred blood cells past the laser beam to stop was recorded.

    ______________________________________                                                                           Positive                                                                Time  or                                         Blood #   Group   Reagent    (s)   Negative*                                  ______________________________________                                        1         A       A            43  +                                                            B           175  -                                          3         A       A            69  +                                                            B           190  -                                          4         B       A          >270  -                                                            B            58  +                                          8         O       A          >200  -                                                            B          >187  -                                          ______________________________________                                         *A positive reaction was defined as Time < 120 s.                        

Agglutination of red blood cells at the leading edge of the blood causedclogging of the track and flow stop. All the blood samples gave theappropriate reaction.

EXAMPLE 5 Blood grouping by flow rate measurements

Materials:

Blood samples as in Example 3; Cartridges (as described in example 2)with 3 μl American Dade blood typing antisera applied (as described inexample 4) to the reagent chamber and dried.

Experimental: Blood (50 μl) was injected into the cartridges at roomtemperature. The time taken for the blood to reach known distances alongthe narrow track was recorded. Flow rates were then calculated from aknowledge of the cross section of the track as a function of distance.

    ______________________________________                                                                             Positive                                 Blood                      Flow Rate at                                                                            or                                       Sample #                                                                              Group    Reagent   150 s(mm.sup.3 /s)                                                                      Negative                                 ______________________________________                                        6       B        A         0.045     -                                                         B         0.025     +                                        1       A        A         0.018     -                                                         B         0.045     +                                        8       O        A         0.047     -                                                         B         0.047     -                                        ______________________________________                                         *A positive reaction was taken as flow rate <0.030 mm.sup.3 /s.          

Agglutination of red blood cells caused a significant reduction in flowrates.

EXAMPLE 6 Use of filters to modify sample composition

a. Red cells were quantitatively removed from whole blood by filtrationthrough a dry filter paper disc impregnated with anti human red cells.Discs were cut to fit the sample application site of cartridges fromBeckman Paragon® electrophoresis blotter paper (about 0.57 mm thick). Ametal punch of diameter 0.467 cm was used. After the discs were snuglyinserted in the sample cite of the track, 10 μl rabbit anti-human redcells (Cappel) was added. This is enough liquid to saturate the paper.The liquid was then evaporated under vacuum.

When blood (40 μl) was applied to the disc about 7 μl clear plasmaemerged into the track before flow stopped.

b. Filter paper disc (as above) impregnated with sodium deoxycholate (10μl×10%) were dried under vacuum and placed in the sample applicationsite. Blood (40 μl) was applied at 37° C. A clear uniform red solutionfree of all red cells filtered into and filled the track. The absorbanceof the remaining hemolysate can give an accurate hemoglobinconcentration.

EXAMPLE 7 Electronic Cartridge

An electronic cartridge capable of simulating the flow of whole bloodthrough a capillary was prepared using a 32768 Hz crystal-controlledoscillator to drive two 1-16th frequency dividers that generated theinput signals for a driver of a liquid crystal display cell prepared inaccordance with the electronic diagram set forth in FIG. 7. This cellwas biased by a 2048 Hz signal modulated at 128 Hz intervals. The celltherefore rotated its polarization at the rate of 128 Hz.

Two more dividers drove a logical AND gate whose output was to a logiclow every 20 seconds. At this point, the output of a logical OR gatereset the dividers, stopping the process. Accordingly, the LCD waspowered and modulating for a 20-second interval.

A start switch was provided to clear the reset signal and restart theprocess. The oscillators, dividers, logic gates, and LCD driver wereimplemented in CMOS technology and powered by a coin-type lithiumbattery. The electronic cartridge has a life of more than 10,000 cycles.

EXAMPLE 8 Out-of-Blood Detector

The following sample depletion device was prepared in the manner shownin FIG. 8. An infrared LED was embedded into the surface of theanalytical device into which a capillary flow cartridge was to beinserted so that the output of its light would impinge upon the bloodreservoir 1 mm from the capillary entrance. The LED is powered by anoperational amplifier configured as a square wave generator of about 8KHz. An infrared-sensitive photodetector was located 2 mm from thejunction of the sample reservoir and the capillary channel. The signaloutput of the detector was passed to a band-pass filter having a centerat 8 KHz and a bandwidth of 500 Hz and was further amplified by anamplifier. The amplified signal was rectified and integrated, therebygenerating a direct current voltage proportional to the scattered light.

When a blood sample is added to the reservoir and flows by capillaryaction through the capillary into other portions of the test cartridge,the light path between the LED and the photodetector is interrupted whenthe reservoir empties. When the voltage output of the integrator fallsbelow 50-30 millivolt, the input of this voltage into a comparator willindicate the absence of blood in the blood reservoir.

EXAMPLE 9 Fabrication

Plastic cartridges were injection molded out of Cycolac® CTB Resin(acrylanitrile-butadiene-styrene copolymer) obtained from Borg-WarnerChemicals, Inc. The design of the cartridge capillary channel andoverall structure is similar to that shown in FIG. 5 in that it containsa sample reservoir, an initial short capillary, a reagent chamber and asecond long capillary. However, the configuration of the actual chambersand capillaries differ from those shown. The cartridge consisted of a30-mil base and a 30-mil cover. Both the cover and base were plasmaetched in an LFE Model 1002 Plasma system with the following settings:argon pressure, 2 torr; argon flow, 1-3; forward RF powere, 100; etchtime, 20 minutes. Three microliters of Biotrack thromboplastin reagentwere then applied to the base of the oval area at the base of thecartridge and allowed to dry for 10 minutes in an environment kept at25° C. and 10% relative humidity. The etched cover was then placed onthe base and welded thereto using a Branson 8400Z ultrasonic welder withthe following settings: down speed, 3 seconds; hold time, 0.5 seconds;weld energy, 0.5 Kjoule; and weld time, 0.26-0.30 second.

Following fabrication, the cartridges were tested with whole bloodcontrols (both abnormal and normal clotting times), which are describedin a copending application filed on even date with this applicationentitled "Whole Blood Control Sample" using the Biotrack Model Monitor1000. The prothrombin times were recorded. In addition, the flow rate ofblood in a cartridge without reagent was measured.

The results of the test were subjected to statistical analysis. Whenreplicates were run, the mean and coefficient of variation were 12.3seconds and 4.9% for the normal control and 20.0 seconds and 2.9% forthe abnormal control. The flow rate of blood in the capillary channelfour days later was constant at 0.06 mm³ /sec. These results are quitesuperior to other fabrication techniques described in thisspecification, including the example using tape set forth in Example 1,which gave C.V.s in the 10-20% range.

All publications and patent applications mentioned in this specificationare indicative of level of skill of those skilled in the art to whichthis invention pertains and are herein incorporated by reference to thesame extent as if each individual patent application and publication hadbeen individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A control device capable of detecting depletionof a particle-containing fluid from a sample reservoir in a devicecomprising a sample reservoir and a capillary exiting said reservoir,which comprises:a light source located so as to impinge on said fluid insaid reservoir; a light detector located in close proximity to saidcapillary so as to collect light which is reflected by said particlesinto said capillary and thereafter further reflected by said particlesso as to pass out through the walls of said capillary; a signalgenerator operably attached to said light source, wherein a detectablesignal is imposed on the output of said light source; and a filteroperably attached to the output of said light detector, wherein saiddetectable signal is isolated from other light sources which may impingeupon said light detector.
 2. The control device of claim 1, wherein saidlight source produces infrared light and said detectable signal is aperiodic variation in the intensity of said light.
 3. The control deviceof claim 1, wherein light from said light source impinges on said fluidin said reservoir at a right angle to a line formed by said capillary.4. The control device of claim 1, wherein said light source is aninfrared light source, said capillary is joined to said reservoir at acapillary entrance, said light source is located from 0.5 to 2 mm fromsaid capillary entrance, and said light detector is located from 1 to 4mm from said capillary entrance.
 5. The control device of claim 1,wherein light from said light source entering said sample reservoir isreflected at essentially a right angle from said light source into saidcapillary and exits said capillary at essentially a right angle toimpinge on said detector.